1. Field of the Invention
The technical field is analytical biochemistry, especially as pertains to diagnostics. More specifically, the invention involves the preparation and use of novel enzyme-based biosensors which blend the useful properties of a tailored dehydrogenase with electrochemiluminescence (ECL) based assay format to detect and the quantify a specific analyte or analytes using conventional biosensor arrangements.
2. Background Information
Biosensors contain biomolecules, such as enzymes or antibodies, or whole-cell testers. Biosensors can be used for rapid, real-time detection of materials in environmental and/or clinical samples. The present biosensor invention involves the creation of enzyme-coenzyme-electrochemiluminescence tagged conjugates. Their development reflects a multidisciplinary effort. The background of the relevant technological areas will be discussed sequentially.
Assays based on electrochemiluminescence (ECL) are well known in the art and are finding expanding applications because of their accuracy, ease of use and freedom from radioactive materials.
A particularly useful ECL system is described in a paper by Yang et al, Bio/Technology, 12, pp. 193-194 (February 1994). See also a paper by Massey, Biomedical Products, October 1992 as well as U.S. Pat. Nos. 5,235,808 and 5,310,687, the contents of these papers and patents being incorporated herein by reference.
ECL processes have been demonstrated for many different molecules by several different mechanisms. In Blackburn et al (1991) Clin.Chem. 37/9, pp. 1534-1539, the authors used the ECL reaction of ruthenium (II) tris(bipyridyl), Ru(bpy)32+, with tripropylamine (TPA) (Leland et al (1990) J. Electrochem. Soc. 137:3127-31) to demonstrate the technique. Salts of Ru(bpy)32+ are very stable, water-soluble compounds that can be chemically modified with reactive groups on one of the bipyridyl ligands to form activated species with which proteins, haptens, and nucleic acids are readily labeled. The activated form of the Ru(bpy)32+ used by Blackburn et al was Ru(bpy)32+-NHS ester: 
The creation of tailored enzyme conjugates is a developing area, especially in the clinical biochemical area. Ebeling et al. (U.S. Pat. No. 5,250,415) utilized recombinant techniques to enhance the quality and stability of NAD/NADP and dependent glucose dehydrogenase (E.C. 1.1.1.42). Through the use of recombinant techniques, isoenzymes were produced in high quality and the enzymes display comparable stability over the range of temperatures between 20° and 50° C. The enzymes are disclosed as suitable for use as medical diagnostic applications.
The creation of semi-synthetic glucose oxidase involving the formation of enzyme conjugates having linked regenerative moieties has been attempted by certain research groups with some success. See Yomo et al., “Preparation and Kinetic Properties of 5-ethylphenazine-glucose-dehydrogenase-NAD+ conjugate, A Semi-Synthetic Glucose Oxidase,” European J. Biochem. (Germany), (Sep. 15, 1991), Vol. 200(3), pp. 759-66. 5-ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-Glc DH-NAD+) was prepared by linking both poly (ethylene glycol)-bound 5-ethyl-phenazine and poly (ethylene glycol)-bound NAD+ to glucose dehydrogenase. This conjugate is a semi-synthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazoyl)-2, 5-diphenyl-2l t-tetra zolium bromide (MTT) as an electron acceptor. The semi-synthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety.
The selective labelling of amino groups in proteins such as beta-D-glucose: NAD(P+) 1-oxidoreductases (EC 1.1.1.47) has been demonstrated. Bozler et al. (Biochim. Biophys. Acta, Vol.749, No. 3, pp. 238-43) selectively labelled glucose dehydrogenase at a lysine group with (2-51-dimethyl amino naphthalene-1-sulfonamido) methylamidic acid methyl ester. The ester was synthesized for the purpose of fluorescence labelling of amino groups of proteins. The incorporation of the dansyl group served as an extrinsic fluorescent probe which can be determined spectrophotometrically.
Biosensors are continually growing area of technology. The term biosensor encompasses a wide range of technologies and reflects multi-disciplinary efforts. There is a continuing need in this area for the enhancement of sensitivity, reliability and rapidity.
A common thread to biosensor technology is the use of a biological material in the measurement of analyte. This is a rather encompassing definition.
A representative text in this field, illustrating the blend of technologies, is Biosensor Design and Application, edited by Paul R. Mathewson and John W. Finley, which was published in 1992 by American Chemical Society, Washington, D.C.
Biosensors, in addition to the requisite biological molecule, can include a variety of measuring systems, e.g. an electrode system comprising a measuring electrode and a luminescence counter wherein the reaction layer contacts the electrode system. The reaction layer typically comprises one molecule electron acceptor, a measuring enzyme and chemiluminescent sensor. The diversity of biosensor systems is evidenced by the devices such as those illustrated in U.S. Pat. Nos. 5,324,835, 5,229,202 and 5,384,028.
Glucose biosensor giving stable measured results without NAD consumption are known. Such biosensors are useful for the qualitative or quantitative determination of one or more components in a liquid mixture by electrochemical regeneration of coenzyme. Typically, such an electrode comprises a redox polymer adsorbed on the surface of the electrode, one or more enzymes wherein at least one is a dehydrogenase and a coenzyme such as NADH, NADPH or their analogues. See Gordon et al, U.S. Pat. No. 5,264,092.
Another approach is evidenced in U.S. Pat. No. 5,340,722 (Wolfbeis et al.) which teaches a continuous and reversible determination using a biosensor which involves a flavin co-enzyme (FMN, FAD), oxidases and oxygenases. The assay is fluorescence based. During enzymatic oxidation the co-enzyme simultaneously transitions into a reduced form, which is then immediately reconverted into the oxidized form by means of oxygen. The transition from oxidized form to reduced form is linked to a change in fluorescence properties, which serve as the analytical parameter.
However, there still remains a need to further enhance the economy, reliability, sensitivity and responsiveness of biosensors.